TY - JOUR
AU - Ikushiro, Shin-ichi
AB - Abstract Mouse UDP-glucuronosyltransferase 1a6 (Ugt1a6) contains two functional copies of 1a6a and 1a6b that share high sequence homology (98%). Only 10 amino acids located around the substrate recognition region are different out of 531 total residues. Although Ugt1a6 plays important roles in conjugating phenolic compounds, the functional characteristics of these isozymes are unclear. We performed functional analyses of mouse Ugt1a6a and Ugt1a6b using two isomeric polyphenols (trans- and cis-resveratrol). The cDNAs of mouse Ugt1a6a and Ugt1a6b were cloned and constructed as recombinant proteins using a yeast expression system, and kinetic parameters were evaluated. The wild-type Ugt1a6a and Ugt1a6b proteins catalysed trans- and cis-resveratrol 3-O-glucuronidation. Although the Km value for trans-resveratrol was significantly lower for Ugt1a6a compared with Ugt1a6b, the Km values for cis-resveratrol were comparable for the isozymes. Despite high sequence homology, significant kinetic differences were observed between the isozymes. To identify the critical residues for resveratrol glucuronidation, we constructed 10 variants of Ugt1a6a (T81P, N96R, H98Q, L100V, S104P, N115S, I117L, V118T, V119L and D120E). The I117L variant had Ugt1a6b-like enzymatic properties of Km in trans-resveratrol, and Vmax and Ksi in cis-form, suggesting that the residues located at position 117 of Ugt1a6a and Ugt1a6b play an important role in resveratrol glucuronidation. cis-resveratrol, kinetics, mouse Ugt1a6a, mouse Ugt1a6b, trans-resveratrol The mammalian UDP-glucuronosyltransferase (UGT) transfers glucuronic acid to various chemicals, such as drugs, natural compounds, steroids, carcinogens and environmental pollutants. The UGT1 and UGT2 families are major enzymes that play important roles in catalysing phase II drug metabolism in mammals. Although understanding inter-species differences in drug metabolism is critical for drug development, animal UGTs remain uncharacterized. Mice are widely used as a rodent pharmacological model for both studies on the early stages of drug development and non-clinical studies. To date, 14 of the Ugt1 genes (including five pseudogenes, ps) and 10 of the Ugt2 genes have been identified in the mouse: Ugt1a1, 1a2, 1a3-ps, 1a4-ps, 1a5, 1a6a, 1a6b, 1a7a-ps, 1a7b-ps, 1a7c, 1a8, 1a9, 1a10, 1a11-ps, 2a1, 2a2, 2a3, 2b1, 2b5, 2b34, 2b35, 2b36, 2b37 and 2b38 (1). Two functional copies of Ugt1a6 (1a6a and 1a6b) have been identified only in mice, and the additional round of duplication/divergence in Ugt1a6 must have occurred after the divergence of rats and mice approximately 17 million years ago (1). Mouse Ugts are distributed in various tissues and are differentially expressed (2). Ugt1a6, which was first cloned from mouse liver by Lamb et al. (3), is mainly localized to the liver, large intestine, lung, stomach and gonad (4). However, the functions of mouse Ugts, including Ugt1a6, for endo- and exobiotic compounds remain unclear. In the present study, we clarify the functional difference between mouse Ugt1a6a and 1a6b. Mouse Ugt1a6a and 1a6b share 98% sequence homology and are identical except for 10 amino acids (http://blast.ncbi.nlm.nih.gov/Blast.cgi; Table I). Among the UGT isoforms, UGT1A6 is the most studied UGT (5). Additionally, UGT1A6 is responsible for conjugating not only simple phenols, such as 1-naphthol or 4-methylumbelliferone (6, 7), but also paracetamol (8) and serotonin (9). However, crystal structures of mammalian UGTs (including UGT1A6) are unavailable except for the co-factor-binding domain of human UGT2B7 (10,11). The binding domain of co-factor UDP-glucuronic acid (UDPGA) is thought to be located at the C-terminal end, which is a highly conserved sequence. In contrast, substrates would bind to the highly variable N-terminal domain (12). Because of the lack of structural knowledge, we attempted to characterize mouse Ugt1a6a and 1a6b by utilizing enzyme kinetics and specific substrates; resveratrol (3,5,4′-trihydroxy-trans-stilbene; Fig. 1A) and its cis-isomer (Fig. 1B), which are dietary polyphenols present in red wine in both their glycosylated and glucuronidated forms (13). In rats, Sabolovic et al. (14) reported that the glucuronidation of resveratrol was stereo- and regio-selective in several tissues and that recombinant rat UGT1A6 and 2B1 were able to glucuronidate trans- and cis-resveratrol. Fig. 1 View largeDownload slide Chemical structures of trans-resveratrol (A) and cis-resveratrol (B). Fig. 1 View largeDownload slide Chemical structures of trans-resveratrol (A) and cis-resveratrol (B). Table I. Comparison of amino acid sequences of mouse and human UGTs. Protein    Amino acid sequence    Mouse Ugt1a6a  81   TYSLEELQTRFRTFGNNHFLPGASLMGPLREYRNNMIVVDM  121  Mouse Ugt1a6b  81   P · · · · · · · · · · · · · ·R ·Q ·V · · ·P · · · · · · · · · ·S ·LTLE ·  121  Human UGT1A6  81   P ·DQ · · ·KN ·YQS · · · · · ·AERSF ·TA ·QT · · · · · · · ·IGL  121  Human UGT1A9  80   S ·T · ·D ·DRE ·KA ·AHAQWKAQVRSIYS ·LMGSY ·D ·FDLF  120  Human UGT1A10  80   S ·T · ·DQNRE ·MV ·AHAQWKAQ ·QSIFS ·LMSSSSGFLDLF  120  Protein    Amino acid sequence    Mouse Ugt1a6a  81   TYSLEELQTRFRTFGNNHFLPGASLMGPLREYRNNMIVVDM  121  Mouse Ugt1a6b  81   P · · · · · · · · · · · · · ·R ·Q ·V · · ·P · · · · · · · · · ·S ·LTLE ·  121  Human UGT1A6  81   P ·DQ · · ·KN ·YQS · · · · · ·AERSF ·TA ·QT · · · · · · · ·IGL  121  Human UGT1A9  80   S ·T · ·D ·DRE ·KA ·AHAQWKAQVRSIYS ·LMGSY ·D ·FDLF  120  Human UGT1A10  80   S ·T · ·DQNRE ·MV ·AHAQWKAQ ·QSIFS ·LMSSSSGFLDLF  120  Ten different residues in Ugt1a6a and 1a6b are showed by bold character. View Large Initially, we cloned Ugt1a6a and 1a6b from major hepatic mouse Ugt isoforms. We also constructed a recombinant mouse Ugt expression system using budding yeast cells to examine the specificity of mouse Ugts for catalysing glucuronidation of trans- and cis-resveratrol. Next, we performed kinetic studies to examine glucuronidation of trans- and cis-resveratrol using recombinant Ugt1a6a and 1a6b. Finally, we constructed amino acid substitution variants of Ugt1a6a to explore the critical amino acids responsible for the functional divergence of Ugt1a6a and 1a6b. Materials and Methods Chemicals and reagents trans-Resveratrol and 7-hydroxycoumarin (umbelliferone) were purchased from Sigma-Aldrich (MO, USA). cis-Resveratrol was purchased from Santa Cruz Biotechnology, Inc. (CA, USA). trans-Resveratrol 3-O-glucuronide, cis-resveratrol 3-O-glucuronide and 7-hydroxycoumarin β-D-glucuronide sodium salt were purchased from Toronto Research Chemicals Inc. (Ontario, Canada). UDPGA was purchased from nacalai tesque Inc. (Kyoto, Japan). Antipeptide and anti-UGT antibodies were previously produced in our laboratory (15,16). Peptide-N-glycosidase F (PNGase F) was purchased from New England Biolabs Inc. (MA, USA). All of the other chemicals and solvents were analytical (or the highest commercially available) grade. cDNA cloning and construction of expression plasmid for mouse Ugt Recombinant mouse Ugts were expressed in budding yeast cells according to previously reported methods (17,18). First, cDNA from mouse Ugts 1a1, 1a5, 1a6a, 1a6b, 1a9 and 2b1 was isolated by reverse transcription–polymerase chain reaction (RT–PCR) from a total RNA fraction prepared from mouse liver using the PCR primer sets shown in Table II. Common primers were used to obtain cDNAs for Ugt1a6a and 1a6b. The pGYR vector was used for the expression of mouse Ugt isoforms; this vector contained a 2 -µm DNA ori, a Leu2 gene as a marker, a pUC ori, Ampr, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter and a terminator derived from Zygosaccharomyces rouxii. To insert the Ugt cDNA into the pGYR vector, the sense primer included a HindIII site upstream of the first ATG, and the antisense primer contained a HindIII site downstream of the stop codon. The amplified full-length DNA was subcloned into the pTA vector (TOYOBO, Osaka, Japan) and sequenced in both directions to confirm each Ugt isoform. Some Ugt isoforms contained internal HindIII sites were altered by point mutations. Finally, HindIII digested fragments were ligated into the HindIII site of the pGYR expression vector. Table II. List of primers used for the construction of mouse Ugt cDNAs. Primer  Sequence (5′–3′)  Ugt1a1 (F)  CCCAAGCTTAAAAAAATGACTGTGGTGTGCTGGAG  Ugt1a5 (F)  CCCAAGCTTAAAAAAATGGGACTCCGCGTGCCCCT  Ugt1a6 (F)  CCCAAGCTTAAAAAAATGGCTTGCCTTCTTCCTGC  Ugt1a9 (F)  CCCAAGCTTAAAAAAATGGTTCCTGCAGCCTTTCC  Ugt2b1 (F)  CCCAAGCTTAAAAAAATGTCTATGAAACAGGCTTC  Ugt1a (R)  CCCAAGCTTTCAATGGGTCTTGGATTGTG  Ugt2b1 (R)  CCCAAGCTTCTACTCTTTTTTCTTCTTTCCCATGT  Primer  Sequence (5′–3′)  Ugt1a1 (F)  CCCAAGCTTAAAAAAATGACTGTGGTGTGCTGGAG  Ugt1a5 (F)  CCCAAGCTTAAAAAAATGGGACTCCGCGTGCCCCT  Ugt1a6 (F)  CCCAAGCTTAAAAAAATGGCTTGCCTTCTTCCTGC  Ugt1a9 (F)  CCCAAGCTTAAAAAAATGGTTCCTGCAGCCTTTCC  Ugt2b1 (F)  CCCAAGCTTAAAAAAATGTCTATGAAACAGGCTTC  Ugt1a (R)  CCCAAGCTTTCAATGGGTCTTGGATTGTG  Ugt2b1 (R)  CCCAAGCTTCTACTCTTTTTTCTTCTTTCCCATGT  F, forward primer; R, reverse primer. View Large Next, we cloned cDNAs of mouse Ugt1a6a variants and constructed their expression plasmids. There are 10 amino acids that differ between mouse Ugt1a6a and 1a6b, and these amino acids of Ugt1a6a were individually substituted into the corresponding location within Ugt1a6b. Thus, we created 10 single-point variants of Ugt1a6a (T81P, N96R, H98Q, L100V, S104P, N115S, I117L, V118T, V119L and D120E). The mutated Ugt1a6a expression plasmids were constructed according to the methods described above, and they utilized the PCR primer sets shown in Table III. Table III. List of primers used for the construction of mouse-Ugt1a6a variant cDNAs. Primer  Sequence (5′ – 3′)  T81P (F)  ATCTTCTCAGTTCCCTACAGCCTAG  T81P (R)  CTAGGCTGTAGGGAACTGAGAAGAT  N96R (F)  GCACCTTTGGACGCAACCACTTTCT  N96R (R)  AGAAAGTGGTTGCGTCCAAAGGTGC  H98Q (F)  TTGGAAACAACCAGTTTCTTCCCGGTGC  H98Q (R)  GCACCGGGAAGAAACTGGTTGTTTCCAA  L100V (F)  AAACAACCACTTTGTTCCCGGTGCCTCC  L100V (R)  GGAGGCACCGGGAACAAAGTGGTTGTTT  S104P (F)  AACCACTTTCTTCCTGGTGCCTCCCTGA  S104P (R)  TCAGGGAGGCACCAGGAAGAAAGTGGTT  N115S (F)  TTCTTCCCGGTGCCACCCTGATGGGTCC  N115S (R)  GGACCCATCAGGGTGGCACCGGGAAGAA  I117L (F)  GAGTACAGGAACAGCATGATTGTCG  I117L (R)  CGACAATCATGCTGTTCCTGTACTC  V118T (F)  ACAACATGATTACCGTGGACATGGA  V118T (R)  AACATGTCCACGGTAATCATGTTGT  V119L (F)  AACATGATTGTCTTGGACATGTTCTTTT  V119L (R)  AAAAGAACATGTCCAAGACAATCATGTT  D120E (F)  GATTGTCGTGGAGATGTTCTTTTCC  D120E (R)  GGAAAAGAACATCTCCACGACAATC  Primer  Sequence (5′ – 3′)  T81P (F)  ATCTTCTCAGTTCCCTACAGCCTAG  T81P (R)  CTAGGCTGTAGGGAACTGAGAAGAT  N96R (F)  GCACCTTTGGACGCAACCACTTTCT  N96R (R)  AGAAAGTGGTTGCGTCCAAAGGTGC  H98Q (F)  TTGGAAACAACCAGTTTCTTCCCGGTGC  H98Q (R)  GCACCGGGAAGAAACTGGTTGTTTCCAA  L100V (F)  AAACAACCACTTTGTTCCCGGTGCCTCC  L100V (R)  GGAGGCACCGGGAACAAAGTGGTTGTTT  S104P (F)  AACCACTTTCTTCCTGGTGCCTCCCTGA  S104P (R)  TCAGGGAGGCACCAGGAAGAAAGTGGTT  N115S (F)  TTCTTCCCGGTGCCACCCTGATGGGTCC  N115S (R)  GGACCCATCAGGGTGGCACCGGGAAGAA  I117L (F)  GAGTACAGGAACAGCATGATTGTCG  I117L (R)  CGACAATCATGCTGTTCCTGTACTC  V118T (F)  ACAACATGATTACCGTGGACATGGA  V118T (R)  AACATGTCCACGGTAATCATGTTGT  V119L (F)  AACATGATTGTCTTGGACATGTTCTTTT  V119L (R)  AAAAGAACATGTCCAAGACAATCATGTT  D120E (F)  GATTGTCGTGGAGATGTTCTTTTCC  D120E (R)  GGAAAAGAACATCTCCACGACAATC  Mutated amino acid codons are underlined. F, forward primer; R, reverse primer. View Large Transformation of Saccharomyces cerevisiae with mouse Ugt plasmids and preparation of microsomal fractions The plasmids (pGYR-mUgts) obtained in the previous section were introduced into S. cerevisiae AH22 cells as described previously (19). The recombinant yeast cells expressing Ugt1a1, 1a5, 1a6a, 1a6b, 1a9, 2b1 and the 10 variants of 1a6a (T81P, N96R, H98Q, L100V, S104P, N115S, I117L, V118T, V119L and D120E) were cultivated in a synthetic, minimal medium containing 2% (w/v) D-glucose and 0.67% (w/v) yeast nitrogen base without amino acids and supplemented with histidine (20 mg/l). The microsomal fractions were prepared as described previously (19). Immunoblot analysis The protein expression level of recombinant Ugt in each microsome was determined by immunoblot analysis. Polyclonal antibodies against a C-terminal peptide, common to both UGT1A and UGT2B isoforms have been developed in our laboratory (15, 16) and were used to detect the mouse Ugts. To obtain clear Ugt bands on the immunoblots, microsomes were treated with PNGase F according to the manufacturer’s protocol and were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) as described previously (20). The proteins in the gel were transferred to nitrocellulose membranes using a semi-dry blotting method. The membranes were blocked with polyvinylidene fluoride (PVDF) Blocking Reagent Can Get Signal® (TOYOBO) or Blocking One (nacalai tesque) at room temperature. The membranes were incubated overnight at room temperature with diluted primary antibodies (anti-Ugt1a antibody; 1: 32 000 or anti-Ugt2b1 antibody; 1: 64 000). Antibodies were diluted with Can Get Signal® solution 1 (TOYOBO) or IMMUNO SHOT reagent 1 (Cosmo Bio Co. Ltd, Tokyo Japan). Blots were then incubated with a diluted anti-primary antibody (1: 4000 dilution) for 4 h at room temperature; the anti-primary antibodies were diluted with Can Get Signal® solution 2 (TOYOBO) or IMMUNO SHOT reagent 2 (Cosmo Bio Co., Ltd.). Anti-primary antibodies were alkaline phosphatase-conjugated goat anti-rabbit IgG (Cell Signaling Technology Inc., MA, USA). Immunodetection was developed by adding a nitro-blue tetrazolium chloride/5-bromo-4-chloro-3′-indolylphosphatase p-toluidine salt (NBT-BCIP) Solution Kit for Alkaline Phosphatase Stain that was purchased from nacalai tesque. The band intensities were quantified using a densitometric scanner and NIH image software (ImageJ, Version 1.38). The relative expression levels of microsomal Ugt1a isoforms as compared with Ugt1a1 were calculated. Glucuronidation assay of trans- and cis-resveratrol in recombinant mouse Ugts The incubation mixture (200 µl total volume) contained 50 mM Tris–HCl buffer (pH 7.5), 8 mM MgCl2, 10 µM of trans-resveratrol or cis-resveratrol and 0.5 mg protein/ml of recombinant mouse Ugts. trans- and cis-Resveratrol were dissolved in dimethyl sulfoxide for a final concentration of 2% (v/v) in the incubation mixtures. After pre-incubating the mixture at 37°C for 5 min, the reactions were initiated by adding UDPGA (2 mM). The reaction mixture was incubated at 37°C for 10 min, and the reaction was terminated by the addition of ice-cold acetonitrile (100 µl). After removing the proteins by centrifugation at 20,000 × g for 20 min at 4°C, aliquots of the supernatants were injected into a high-performance liquid chromatography (HPLC) with the Wakopak Navi C30-5 analytical column (3.0 × 150 mm, Wako Pure Chemical Ind., Ltd., Osaka, Japan) at 40°C. The mobile phase A was distilled water, and the mobile phase B was 0.5% (v/v) formic acid/acetonitrile. The HPLC was operated in an isocratic mode with a flow rate of 1.0 ml/min, and the mobile phases A (85%) and B (15%) were mixed in a liquid chromatography (LC) pump for the analysis of trans-resveratrol and its 3-O-glucuronide and monitored at 303 nm. For the analysis of cis-resveratrol and its 3-O-glucuronide, monitoring was performed at 286 nm, and the following step gradient was used: hold at 10% B for 5 min, hold at 15% B for 35 min and hold at 10% B for 5 min with a flow rate of 1.0 ml/min. The quantification ranges for 3-O-glucuronides were 0.1–400 µM for the trans-form and 0.5–100 µM for the cis-form with each standard. Glucuronidation assay for Ugt1a6a variants using trans-resveratrol and 7-hydroxycoumarin The 10 recombinant mouse Ugt1a6a variants were screened to find the Ugt1a6b-type variant using trans-resveratrol and 7-hydroxycoumarin, which is a universal substrate for Ugt isoforms (21). The incubation and preparation of analytical samples were performed under the same conditions described above with 10 µM and 50 µM of trans-resveratrol or 1 mM of 7-hydroxycoumarin. In the assay utilizing 7-hydroxycoumarin, the microsome concentration was 1.0 mg protein/ml, and the incubation time was 120 min. Ultra-HPLC analysis was used for samples using 7-hydroxycoumarin, and the analytical column was the COSMOSIL PACKED COLUMN 2.5 C18-MS-II (2.0 × 100 mm, nacalai tesque Inc.) held at 45°C. The mobile phase A was 0.1% trifluoroacetic acid, and the mobile phase B was 0.1% trifluoroacetic acid/acetonitrile. The ultra-HPLC was operated in the linear gradient mode and employed the following settings: hold at 10% B for 4 min, 10–60% B for 4 min and 60–10% B for 2 min with a flow rate of 0.5 ml/min. 7-Hydroxycoumarin and its glucuronide were monitored at 320 nm and glucuronides were quantitated with a concentration range of 5–100 µM with the standard. The activity ratio of trans-resveratrol glucuronidation was then calculated by the following equation: activity ratio = velocity (nmol/min/mg) with 50 µM substrate/velocity with 10 µM substrate. This activity ratio and the glucuronidation activity of 7-hydroxycoumarin (normalized with the relative Ugt expression level) were used to distinguish between the Ugt1a6b-type and Ugt1a6a-type variants. From the results of the screening study, the Ugt1a6b-type variant was selected and further studies related to the kinetics for trans- and cis-resveratrol were conducted. Kinetic study of trans- and cis-resveratrol in Ugt1a6a, 1a6b and Ugt1a6a variants The incubation mixture was constructed with the same components described in the previous section with 1–500 µM of trans-resveratrol or 1–100 µM of cis-resveratrol and 0.5 mg protein/ml of recombinant mouse Ugt1a6a, 1a6b and Ugt1a6a variants (S104P and I117L). After pre-incubating the mixture at 37°C for 5 min, the reactions were initiated by adding UDPGA (2 mM), and the reaction mixture was incubated at 37°C for 2–60 min such that the conditions produced glucuronidation velocities in the linear range. The reaction was terminated, and proteins were removed by centrifugation (as described above) and subjected to HPLC analysis. Kinetic data analysis Kinetic parameters were estimated from the fitted curves using the Kaleida Graph computer program (Synergy Software, PA, USA), which was designed for nonlinear regression analysis. The following equation was applied for substrate inhibition kinetics (equation 1):   (1) where V is the rate of the reaction, Vmax is the maximum velocity, Km is the Michaelis constant, S is the substrate concentration, and Ksi is the constant describing the substrate inhibition interaction (22). Other methods The protein concentrations of the recombinant microsomes were determined with the bicinchoninic acid (BCA) protein assay reagent (nacalai tesque) using bovine serum albumin as the standard. Results Protein expression of recombinant mouse Ugt isoforms The protein expression levels of the recombinant Ugt1a and 2b1 in yeast microsomes were determined using immunoblot analysis with specific anti-peptide antibodies (Fig. 2). Anti-Ugt1a antibody commonly binds to a C-terminal region of all Ugt1a proteins, and the expression levels were confirmed to be comparable among the many isoforms in yeast microsomes. Fig. 2 View largeDownload slide Immunoblot analysis of mouse Ugt1a and Ugt2b1 in yeast microsomes. Microsomes containing 10 -µg protein were used in the immunoblot analyses. Values under the panel represent the mean relative expression level of Ugt1a (the expression level of Ugt1a1 = 1.0) in yeast microsomes (n = 4). Fig. 2 View largeDownload slide Immunoblot analysis of mouse Ugt1a and Ugt2b1 in yeast microsomes. Microsomes containing 10 -µg protein were used in the immunoblot analyses. Values under the panel represent the mean relative expression level of Ugt1a (the expression level of Ugt1a1 = 1.0) in yeast microsomes (n = 4). Glucuronidation activity of recombinant mouse Ugt isoforms for trans- and cis-resveratrol Chromatograms of trans- and cis-resveratrol and their glucuronides with microsomes of Ugt1a6a and 1a6b are shown (Fig. 3). Ugt1a1, 1a6a and 1a6b showed higher glucuronidation activity (Fig. 4) for both trans- and cis-resveratrol (10 µM or 100 µM). The 3-O-glucuronidation by Ugt1a6a and 1a6b was detected for both of the isomers (trans-form at 5 min, Fig. 3A and cis-form at 16 min, Fig. 3B). 4′-O-glucuronide of trans- or cis-resveratrol was not detected for all Ugt isoforms tested (data not shown); if present, these glucuronides should have been observed at 3 min and 14 min, respectively. Fig. 3 View largeDownload slide HPLC profiles of resveratrol glucuronide. (A) trans-Resveratrol (10 µM) was incubated at 37°C with UDPGA in recombinant Ugt1a6a (10 min) or 1a6b (60 min); (B), cis-Resveratrol (10 µM) was incubated at 37°C with UDPGA for 10 min in recombinant Ugt1a6a or 1a6b. Fig. 3 View largeDownload slide HPLC profiles of resveratrol glucuronide. (A) trans-Resveratrol (10 µM) was incubated at 37°C with UDPGA in recombinant Ugt1a6a (10 min) or 1a6b (60 min); (B), cis-Resveratrol (10 µM) was incubated at 37°C with UDPGA for 10 min in recombinant Ugt1a6a or 1a6b. Fig. 4 View largeDownload slide 3-O-glucuronidation activity of trans-resveratrol (A) and cis-resveratrol (B) in recombinant mouse Ugts. Resveratrol was incubated at 10 µM (open bar) or 100 µM (closed bar) for 10 min at 37°C with 2 mM UDPGA and 0.5 mg protein/ml of microsome. Each value represents mean ± SD (n = 3). Fig. 4 View largeDownload slide 3-O-glucuronidation activity of trans-resveratrol (A) and cis-resveratrol (B) in recombinant mouse Ugts. Resveratrol was incubated at 10 µM (open bar) or 100 µM (closed bar) for 10 min at 37°C with 2 mM UDPGA and 0.5 mg protein/ml of microsome. Each value represents mean ± SD (n = 3). Kinetics of Ugt1a6a and 1a6b-dependent glucuronidation of trans- and cis-resveratrol Kinetic analysis of trans- and cis-resveratrol glucuronidation was performed using recombinant mouse Ugt1a6a and 1a6b. The data points were fitted to the substrate-inhibition equation (equation 1) to yield the kinetic parameters (Fig. 5 and Table IV). For trans-resveratrol, Ugt1a6a exhibited a significantly lower Km value (3.20 µM) than that of Ugt1a6b (376 µM). The Vmax/Km value for Ugt1a6a was 40-fold higher than that for Ugt1a6b. In contrast, the Km values for cis-resveratrol were comparable between Ugt1a6a (1.81 µM) and 1a6b (1.62 µM); the Vmax/Km values for each isozyme were also comparable. These results indicate that Ugt1a6a and 1a6b are functionally different and that trans-resveratrol is a better substrate for distinguishing enzymatic properties between Ugt1a6a and 1a6b. Fig. 5 View largeDownload slide Kinetics for 3-O-glucuronidation of trans-resveratrol (A) and cis-resveratrol (B) by recombinant Ugt1a6a (open circle), Ugt1a6b (open square) and mutated Ugt1a6a (I117L, open diamond). Each value (n = 3, mean ± SD) and fitting curve (solid line, WT; dashed line, variant) are plotted. trans-Resveratrol (1–500 µM) was incubated for 2–60 min at 37°C. cis-Resveratrol (1–100 µM) was incubated for 2–30 min at 37°C. Fig. 5 View largeDownload slide Kinetics for 3-O-glucuronidation of trans-resveratrol (A) and cis-resveratrol (B) by recombinant Ugt1a6a (open circle), Ugt1a6b (open square) and mutated Ugt1a6a (I117L, open diamond). Each value (n = 3, mean ± SD) and fitting curve (solid line, WT; dashed line, variant) are plotted. trans-Resveratrol (1–500 µM) was incubated for 2–60 min at 37°C. cis-Resveratrol (1–100 µM) was incubated for 2–30 min at 37°C. Table IV. Kinetic properties of WT Ugt1a6a, 1a6b and Ugt1a6a variants with trans- and cis-resveratrol. Isomer  Ugt  Vmax (nmol/min/mg protein)  Km (μM)  Ksi (μM)  Vmax / Km (ml/min/mg protein)  trans-  1a6a  1.42 ± 0.14  3.20 ± 1.12  517 ± 220  0.444  1a6b  4.16 ± 1.38  376 ± 149  175 ± 79  0.011  1a6a I117L  2.00 ± 0.19  55.0 ± 9.0  420 ± 87  0.036  1a6a S104P  1.38 ± 0.09  5.81 ± 0.96  245 ± 44  0.238  cis-  1a6a  2.34 ± 0.12  1.81 ± 0.31  515 ± 241  1.293  1a6b  5.15 ± 0.52  1.62 ± 0.55  141 ± 55  3.179  1a6a I117L  5.83 ± 0.32  2.93 ± 0.42  131 ± 25  1.990  1a6a S104P  1.75 ± 0.11  0.939 ± 0.260  1070 ± 1260  1.864  Isomer  Ugt  Vmax (nmol/min/mg protein)  Km (μM)  Ksi (μM)  Vmax / Km (ml/min/mg protein)  trans-  1a6a  1.42 ± 0.14  3.20 ± 1.12  517 ± 220  0.444  1a6b  4.16 ± 1.38  376 ± 149  175 ± 79  0.011  1a6a I117L  2.00 ± 0.19  55.0 ± 9.0  420 ± 87  0.036  1a6a S104P  1.38 ± 0.09  5.81 ± 0.96  245 ± 44  0.238  cis-  1a6a  2.34 ± 0.12  1.81 ± 0.31  515 ± 241  1.293  1a6b  5.15 ± 0.52  1.62 ± 0.55  141 ± 55  3.179  1a6a I117L  5.83 ± 0.32  2.93 ± 0.42  131 ± 25  1.990  1a6a S104P  1.75 ± 0.11  0.939 ± 0.260  1070 ± 1260  1.864  Each value represents a best-fit value ± computer-calculated standard error of triplicate points. View Large Glucuronidation activities of Ugt1a6a variants for trans-resveratrol and 7-hydroxycoumarin To explore the critical residues characterizing Ugt1a6a and 1a6b activity, single amino acid substitution variants of mouse Ugt1a6a (T81P, N96R, H98Q, L100V, S104P, N115S, I117L, V118T, V119L and D120E) were constructed, and corresponding yeast microsomes were obtained. The protein expression level of Ugt1a in each variant microsome was determined by immunoblot analysis (Fig. 6). The expressions of variant T81P were significantly lower than those of other variants. The protein band is almost invisible but the integrated values obtained by ImageJ software were 2- to 60-fold higher than the background values. The low expression of the variant confirmed in four independent analyses. To screen the substitution variants with Ugt1a6b-type properties, the glucuronidation activities of the variants for trans-resveratrol were normalized by the Ugt expression level. Glucuronidation activities were evaluated with two concentrations of trans-resveratrol (10 µM and 50 µM). Several Ugt1a6a variants showed comparable activities with that of wild-type (WT) Ugt1a6a. In contrast, the activities were increased for the H98Q variant and were decreased in the L100V, V118T and D120E variants (Fig. 7A). Next, the activity ratios (v[S] = 50 µM/v[S] = 10 µM) were calculated to highlight the Ugt1a6b-type variant properties. Reflecting the kinetic properties, the activity ratio in WT Ugt1a6b was significantly higher than that of WT Ugt1a6a. Among the 10 variants, the I117L variant had a significantly higher activity ratio than the other variants, and the ratio was comparable with that of WT Ugt1a6b. These results indicate that the I117L Ugt1a6a variant has Ugt1a6b-like properties (Fig. 7B). Fig. 6 View largeDownload slide Protein expression of WT Ugt1a6a, 1a6b and Ugt1a6a variants. Microsomes containing 10 -µg protein were used in the immunoblot analyses. Values under the panel represent the mean relative expression level of Ugt1a (the epression level of Ugt1a6a WT = 1.0) in recombinant microsomes (n = 4). Fig. 6 View largeDownload slide Protein expression of WT Ugt1a6a, 1a6b and Ugt1a6a variants. Microsomes containing 10 -µg protein were used in the immunoblot analyses. Values under the panel represent the mean relative expression level of Ugt1a (the epression level of Ugt1a6a WT = 1.0) in recombinant microsomes (n = 4). Fig. 7 View largeDownload slide 3-O-glucuronidation activity and its activity ratio of trans-resveratrol in recombinant WT Ugt1a6b, WT Ugt1a6a and its variants. (A) 3-O-glucuronidation activity of trans-resveratrol (10 µM, open bar or 50 µM, closed bar) in microsomes of WT Ugt1a6b, WT Ugt1a6a and its variants; (B) activity ratio calculated by velocity values of trans-resveratrol 3-O-glucuronidation (v[S] = 50 µM/v[S] = 10 µM). trans-Resveatrol was incubated for 10 min at 37°C with microsomes. The velocity value was normalized with the relative expression level of Ugt1a6 in each microsome. Each value represents mean ± SD (n = 3). Fig. 7 View largeDownload slide 3-O-glucuronidation activity and its activity ratio of trans-resveratrol in recombinant WT Ugt1a6b, WT Ugt1a6a and its variants. (A) 3-O-glucuronidation activity of trans-resveratrol (10 µM, open bar or 50 µM, closed bar) in microsomes of WT Ugt1a6b, WT Ugt1a6a and its variants; (B) activity ratio calculated by velocity values of trans-resveratrol 3-O-glucuronidation (v[S] = 50 µM/v[S] = 10 µM). trans-Resveatrol was incubated for 10 min at 37°C with microsomes. The velocity value was normalized with the relative expression level of Ugt1a6 in each microsome. Each value represents mean ± SD (n = 3). Furthermore, the glucuronidation activities of Ugt1a6a variants for 7-hydroxycoumarin was assessed. The glucuronidation activity of WT Ugt1a6b for 7-hydroxycoumarin was 2.4-fold higher than the activity of WT Ugt1a6a. Among the 10 variants, the activity profiles for glucuronidation of 7-hydroxycoumarin were similar to those for trans-resveratrol; L100V, V118T and D120E variants exhibited lower activity. The I117L variant showed significantly higher activity than WT Ugt1a6a, and this activity was comparable with that of WT Ugt1a6b (Fig. 8). These results strongly suggest that the amino acid residues at position 117 of Ugt1a6a and Ugt1a6b play an important role in their enzymatic property for glucuronidation. Fig. 8 View largeDownload slide 7-O-glucuronidation activity of hydroxycoumarin in recombinant WT Ugt1a6b, WT Ugt1a6a and its variants. 7-Hydroxycoumarin (1 mM) was incubated for 120 min at 37°C with microsomes. The velocity value was normalized with the relative expression level of Ugt1a6 in each microsome. Each value represents mean ± SD (n = 3). Fig. 8 View largeDownload slide 7-O-glucuronidation activity of hydroxycoumarin in recombinant WT Ugt1a6b, WT Ugt1a6a and its variants. 7-Hydroxycoumarin (1 mM) was incubated for 120 min at 37°C with microsomes. The velocity value was normalized with the relative expression level of Ugt1a6 in each microsome. Each value represents mean ± SD (n = 3). Kinetics of Ugt1a6a variant-dependent glucuronidation for trans- and cis-resveratrol From the results of the screening study, the I117L variant was subjected to kinetic studies with trans- and cis-resveratrol. The kinetic study showed that the I117L mutation within Ugt1a6a altered many parameters, and the variant behaved similarly to Ugt1a6b with respect to Km (55.0 µM) and Vmax/Km with trans-resveratrol and with respect to Vmax (5.83 nmol/min/mg protein) with cis-resveratrol (Fig. 5 and Table IV). Conversely, the S104P variant (negative control) did not affect the kinetics for either trans- (Km = 5.81 µM and Vmax = 1.38 nmol/min/mg protein) or cis-resveratrol (Km = 0.939 µM and Vmax = 1.75 nmol/min/mg protein). The negative control also exhibited the same kinetic pattern as that of WT Ugt1a6a (Table IV). These results indicate that Ile117 of Ugt1a6a and Leu117 of Ugt1a6b are key amino acids that help to characterize the activity of each isoform and that these amino acids play an important role in catalysing the glucuronidation of resveratrol. Discussion We performed functional analyses of mouse Ugt1a6a and 1a6b, which share 98% amino acid sequence homology. Mouse Ugt1a6a and 1a6b consist of 531 amino acids, and only 10 amino acids located around the substrate recognition region in the N-terminal portion of the protein vary between Ugt1a6a and 1a6b. However, previous studies have investigated their expression levels or functions as ‘formal Ugt1a6’ and not separately as Ugt1a6a and Ugt1a6b. This report is the first to reveal the functional difference in the enzymatic properties of mouse Ugt1a6a and 1a6b; this difference was distinguished by using stereoisomeric polyphenols (trans- and cis-resveratrol) as model substrates. The main results obtained in the present study are twofold. First, mouse Ugt1a6a exhibited a significantly lower Km value for trans-resveratrol glucuronidation when compared with Ugt1a6b, and the Km values of the two isoforms were similar for cis-resveratrol glucuronidation. Second, the I117L single-point variant of Ugt1a6a behaved like Ugt1a6b with respect to the glucuronidation kinetics for trans- and cis-resveratrol. Initially, we performed a pilot study for trans- and cis-resveratrol glucuronidation with recombinant mouse microsomal Ugt1a1, 1a5, 1a6a, 1a6b, 1a9 and 2b1, which are major hepatic Ugts (4) obtained from yeast cells. Ugt1a1, 1a6a and 1a6b were capable of trans- and cis-resveratrol glucuronidation, and only 3-O-glucuronides were formed with both isomers. In the literature, 4′-O-glucuronide of trans-resveratrol has not been observed in mice (23). For cis-resveratrol, limited data for other species are available, but 4′-O-glucuronide was not observed in recombinant human UGT1A6 (24, 25). Additionally, rat UGT1A6 exhibited much lower activity of 4′-O-glucuronidation than that of 3-O-glucuronidation (14). Therefore, we focused on 3-O-glucuronide formation and denoted it as ‘glucuronide’ in this report. Second, the kinetics of trans- and cis-resveratrol glucuronidation by recombinant mouse Ugt1a6a and 1a6b were evaluated. All of the plots obtained were fitted to the substrate inhibition model to estimate the kinetic parameters with high precision. Several studies in the literature have reported atypical kinetics of trans-resveratrol (23, 26) and cis-resveratrol (27) utilizing various protein sources. For trans-resveratrol, significantly different kinetics were observed between Ugt1a6a and 1a6b, and Ugt1a6a had a much lower Km value. However, the Km values were comparable between Ugt1a6a and 1a6b with respect to cis-resveratrol, and the Vmax value was higher for Ugt1a6b. These results indicate that Ugt1a6a and 1a6b are functionally different and that trans-resveratrol is a better substrate for distinguishing this functional difference. Next, we constructed variants of mouse Ugt1a6a to identify critical residues that are responsible for the different enzymatic activities of Ugt1a6a and Ugt1a6b. Comparing the amino acid sequences of mouse Ugt1a6a and 1a6b, only 10 amino acids are different. We substituted each of these 10 residues of Ugt1a6a individually into the corresponding location of Ugt1a6b (T81P, N96R, H98Q, L100V, S104P, N115S, I117L, V118T, V119L and D120E). These residues are located around the substrate recognition region and are proposed to reside in the N-terminal half of the protein (12, 28–31). The residues Asn115, Ile117, Val118, Val119 and Asp120 might reside within an α-helix motif based upon the secondary structure of mouse Ugt1a6a as predicted by PSIPRED v3.0 (http://bioinf.cs.ucl.ac.uk/psipred/). Although the glucuronidation activities varied greatly among the individual substitution variants, Ugt expression levels were detected (Fig. 6). The protein expression level of variant T81P was significantly low (2- to 60-fold higher than the background values). The reason for this low level of expression is unclear, but glucuronidation activities for trans-resveratrol and 7-hydroxycoumarin were observed with the T81P variant, suggesting that a trace amount of protein was expressed. Reflecting the kinetics pattern, the activity ratio (v[S] = 50 µM/v[S] = 10 µM) of trans-resveratrol glucuronidation was higher (2.1-fold) in WT Ugt1a6b than in WT Ugt1a6a. This activity ratio was used for screening an Ugt1a6b-like substitution variant. Additionally, 7-hydroxycoumarin (a universal UGT substrate) was used to determine the reliability of the screening result. Interestingly, the I117L variant of Ugt1a6a showed a significantly higher activity ratio for trans-resveratrol glucuronidation and a higher activity for 7-hydroxycoumarin glucuronidation when compared with the other variants. These results strongly indicate that Ile117 and Leu117 are the critical residues distinguishing enzymatic activities of Ugt1a6a and 1a6b. Although the V119L variant showed a somewhat higher activity ratio than WT Ugt1a6a (Fig. 7B), its 7-hydroxycoumarin glucuronidation activity was significantly lower than that of the I117L variant and WT Ugt1a6b (Fig. 8); these results suggest that the V119L variant retained the wild-type activities. It should be mentioned that the V118T and D120E mutations dramatically decreased the glucuronidation of trans-resveratrol and 7-hydroxycoumarin. The region from Ile117 to Asp120 may be critical for the glucuronidation activity of Ugt1a6a. Following the screening results, a kinetics study was conducted with the I117L variant of Ugt1a6a. The Km value of the I117L variant for trans-resveratrol and the Vmax and Ksi values for cis-resveratrol resembled those of WT Ugt1a6b. These results suggest that the 117th residue of mouse Ugt1a6a and 1a6b is critical for the binding affinity (Km) in trans-resveratrol, and capacity (Vmax) and/or binding affinity under the substrate-inhibition state (Ksi) in cis-resveratrol. Ile and Leu are branched amino acids, and these structural isomers have hydrophobic side chains. Surprisingly, a single substitution of Ile for Leu determines the enzymatic properties of Ugt1a6a and 1a6b. In another example, a single-point mutation of Leu to Ile at the HIV-1 reverse transcriptase codon 74 affected its processivity under the presence of another mutation (32). In mouse Ugt1a6, the Ile117 and Leu117 may be located near the substrate recognition region and might be a part of the substrate binding pocket orientating the hydrophobic side chain into the inside of the pocket. The substitution of Ile to Leu may alter the spatial variability of the substrate-binding pocket and may elevate the Km value for trans-resveratrol glucuronidation. This change in the spatial variability may have minor effects on the binding of cis-resveratrol. Conversely, the S104P variant did not change the glucuronidation kinetics for either trans- or cis-resveratrol, even though Ser likely interacts with the substrate, and Pro would likely disrupt the tertiary structure of the protein. Itäaho et al. (33) reported that Phe117 of human UGT1A9 may participate in 1-naphthol binding. Human UGT1A10 contains Leu117, and the F117L mutation of UGT1A9 changed the kinetic model from bi-phasic to substrate inhibition; the Km value resembled that of UGT1A10. Furthermore, their docking model of UGT1A9 and 1-naphthol showed that 1-naphthol docked between the catalytic His37 and the anomeric carbon of the glucuronic acid moiety of UDPGA; 1-naphthol is also capable of contacting Phe117 because of its rigidity and rotational capacity. The homologies among mouse Ugt1a6 and known human UGTs are low (Table I). However, a human UGT1A9 model suggested by Itäaho was constructed based on their human UGT1A1 model (34), and its secondary structure is similar to our secondary structures for mouse Ugt1a6a and 1a6b, which were predicted with PSIPRED v3.0 (http://bioinf.cs.ucl.ac.uk/psipred/). Given the hypothesis that the tertiary structures of human UGT1A9 and mouse Ugt1a6 are similar, the 117th residue (and residues nearby) of mouse Ugt1a6a (Ile) and 1a6b (Leu) could play an important role as a component of the substrate-binding pocket. Guo et al. (35) compared the haplotypes of mouse Ugt1a among 15 mouse strains and found that 9 SNPs altered the amino acid sequence located within the domain responsible for catalysing the transfer of UDPGA to a substrate. These SNPs in mouse strains were found only in Ugt1a9 or 1a7c and they did not change the mRNA levels, whereas SN-38 glucuronidation activity was significantly affected by the SNPs. According to Guo et al., Ugt1a6 has no SNPs in any of the mouse strains, and mRNA expression levels varied throughout the different mouse strains. Therefore, we could disregard the influence of SNPs for Ugt1a6a and 1a6b. Shiratani et al.(36) found significant differences among mouse strains with respect to glucuronidation activities of 7-hydroxy-4-methylcoumarin (4-methylumbelliferone) and 4-nitrophenol, which are substrates of Ugt1a6. Buckley and Klaassen (37, 38) suggested that mouse Ugt1a6 was regulated by several nuclear receptors and found that gender differences in mouse Ugt mRNA were influenced by male-pattern growth hormones. We speculate that expression levels of Ugt1a6a and 1a6b may be different between male and female mice and they may vary among strains, leading to gender and/or strain differences in Ugt1a6 activity. A limitation of our study is that protein expression levels of each Ugt1a6a and 1a6b in various mouse tissues have not been verified, and further study is needed to clarify the expression of Ugt1a6a and 1a6b in mouse tissues. In conclusion, of the 10 amino acids that differ between mouse Ugt1a6a and 1a6b, the 117th residue determines the important properties of mouse Ugt1a6a and 1a6b such as affinity or capacity in catalysing trans- and cis-resveratrol glucuronidation. We revealed the functional differences in the enzymatic properties of mouse Ugt1a6a and Ugt1a6b. Acknowledgements The authors are grateful to Ms Yuka Masuyama, Ms Natsuki Hioki and Ms Nanami Nishiguchi for the construction of recombinant mouse Ugt and for experimental support. Conflict of interest None declared. 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Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
TI - The critical role of amino acid residue at position 117 of mouse UDP-glucuronosyltransfererase 1a6a and 1a6b in resveratrol glucuronidation
JF - The Journal of Biochemistry
DO - 10.1093/jb/mvs078
DA - 2012-07-05
UR - https://www.deepdyve.com/lp/oxford-university-press/the-critical-role-of-amino-acid-residue-at-position-117-of-mouse-udp-09O5NN00Ea
SP - 331
EP - 340
VL - 152
IS - 4
DP - DeepDyve
ER -